Optical fiber microscopy launch system and method

ABSTRACT

A launch system and method for microscopy having an optical fiber positioned proximate a sample slide with an optical fiber mounting element so as to deliver an EMR from the optical fiber into a first sample slide and to a surface of a second sample slide at a critical angle for total internal reflection at an interface of the surface of the second sample slide and a sample positioned proximate to the surface of the second sample slide.

RELATED APPLICATION DATA

This application is a continuation application of U.S. patentapplication Ser. No. 11/441,360, filed May 25, 2006, and titled “OpticalFiber Microscopy Launch System and Method” (now U.S. Pat. No. 7,433,563,issued Oct. 7, 2008), which is incorporated by reference herein in itsentirety. This application also claims the benefit of priority of U.S.Provisional Patent Application Ser. No. 60/684,465, filed May 25, 2005,and titled “Total Internal Reflection System and Method Using OpticalFiber,” which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to the field of microscopy. Inparticular, the present invention is directed to an optical fibermicroscopy launch system and method.

BACKGROUND

Microscopes vary is size and complexity. Typically, the delivery ofexcitation and/or illumination radiation to a sample in a microscopysystem requires complicated optical components that direct and redirectthe radiation from a source through the microscope system to the sample.Translation of the sample (e.g., for focusing the sample to an objectivelens of the microscope) can interfere with the positioning of thedelivered radiation. In several applications the angle of delivery ofthe radiation to the sample and/or a sample slide are critical to theresults of the microscopy method. When the sample platform is moved withrelation to the radiation delivery mechanisms, the delivery mechanismsmay require readjustment to ensure the proper delivery of the radiationto the sample. Further, modifying an existing microscope to performcertain specific microscopy methods (e.g., total internal reflection(TIR) microscopy) can be a costly and complex process.

Total internal reflection (TIR) occurs when electromagnetic radiation(EMR), typically light, strikes an interface between two optical mediaat an incident angle equal to, or greater than the critical angle. FIG.1 illustrates one example of TIR at an interface 110 between a glassmicroscope sample slide 120 and a sample 130. The incoming beam ofelectromagnetic radiation (EMR) 140 is incident at interface 110 at acritical angle 150. TIR energy 160 is reflected, and an evanescent field170 is thereby produced and emanates into sample 130. The critical anglefor TIR at an interface of a first and second material can be given as:θ_(c)=sin⁻¹(n ₂ /n ₁), where n ₁ >n ₂,where n₁ is the refractive index of the first material, n₂ is therefractive index of the second material, and θ_(c) is the criticalangle.

SUMMARY OF THE DISCLOSURE

In one embodiment, a launch system for total internal reflectionmicroscopy is provided. The system includes a first sample slide havinga first side and a second side; an optical fiber mounting elementpositioned in optical contact with said first side; a second sampleslide having a third side and a fourth side; and an optical fiber havinga first terminal end with a terminal surface, said optical fiber beingoptically coupled with said optical fiber mounting element so as toposition said optical fiber to deliver an electromagnetic radiation fromsaid terminal end through said first side into said first sample slide,such that when said second side and said third side are brought intooptical contact and a sample is positioned proximate said fourth side,said electromagnetic radiation is delivered to said fourth side at anincident angle that is at least a critical angle for total internalreflection at an interface of said fourth side and the sample.

In another embodiment, a launch system for total internal reflectionmicroscopy is provided. The system includes a first sample slide havinga first side, a second side, and a fiber insertion portal in said firstside; and a second sample slide having a third side and a fourth side,said fiber insertion portal having a fiber insertion axis, the fiberinsertion axis configured such that when said second side and said thirdside are brought into optical contact and an optical fiber is insertedwithin said fiber insertion portal, the optical fiber is positioned soto deliver an electromagnetic radiation to said fourth side at anincident angle, such that when a sample is positioned proximate saidfourth side, said incident angle is at least a critical angle for totalinternal reflection at an interface of said fourth side and the sample.

In yet another embodiment, a total internal reflection microscopy launchsystem for delivering an electromagnetic radiation to a first sampleslide having a first side and a second side and to a second sample slidehaving a third side and a fourth side is provided. The system includesan optical fiber mounting element configured to be positioned in opticalcontact with the first side; and an optical fiber having a firstterminal end having a terminal surface, said optical fiber beingoptically coupled with said optical fiber mounting element so as toposition said optical fiber to deliver an electromagnetic radiation fromsaid terminal end through said first side into said first sample slide,such that when said second side and said third side are brought intooptical contact and a sample is positioned proximate said fourth side,said electromagnetic radiation is delivered to said fourth side at anincident angle that is at least a critical angle for total internalreflection at an interface of said fourth side and the sample.

In still another embodiment, a method of performing total internalreflection microscopy is provided. The method includes providing a firstsample slide having a first side and a second side; positioning anoptical fiber mounting element in optical contact with said first side;providing a second sample slide having a third side and a fourth side;positioning a sample proximate said fourth side; positioning said secondside in optical contact with said third side; and optically coupling anoptical fiber with the optical fiber mounting element so as to positionthe optical fiber to deliver an electromagnetic radiation from theoptical fiber through said first side into said first sample slide andinto said second sample slide to said fourth side at an incident anglethat is at least a critical angle for total internal reflection at aninterface of said fourth side and the sample.

In still yet another embodiment, a method of modifying a first sampleslide for total internal reflection microscopy is provided. The firstsample slide includes a first side and a second side. The methodincludes positioning an optical fiber at a first position that isproximate the first side so as to position the optical fiber to deliveran electromagnetic radiation into the first sample slide such that whena second sample slide having a third side and a fourth side ispositioned with the third side in optical contact with the second sideand a sample is positioned proximate the fourth side, theelectromagnetic radiation is delivered into the second sample slide tothe fourth side at an incident angle that is at least a critical anglefor total internal reflection at an interface of the fourth side and thesample.

In a further embodiment, a method of modifying a microscope for a totalinternal reflection microscopy technique is provided. The methodincludes providing a first sample slide having a first side and a secondside; positioning an optical fiber mounting element in optical contactwith said first side; providing a second sample slide having a thirdside and a fourth side; positioning a sample proximate said fourth side;positioning said second side in optical contact with said third side;and optically coupling an optical fiber with the optical fiber mountingelement so as to position the optical fiber to deliver anelectromagnetic radiation from the optical fiber through said first sideinto said first sample slide and into said second sample slide to saidfourth side at an incident angle that is at least a critical angle fortotal internal reflection at an interface of said fourth side and thesample.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, the drawings show aspectsof one or more embodiments of the invention. However, it should beunderstood that the present invention is not limited to the precisearrangements and instrumentalities shown in the drawings, wherein:

FIG. 1 shows one example of total internal reflection;

FIG. 2 shows one embodiment of an optical fiber microscopy launchsystem;

FIG. 3 shows another embodiment of an optical fiber microscopy system;

FIG. 4 shows one embodiment of an optical fiber mounting element;

FIG. 5 shows another embodiment of an optical fiber mounting element;

FIG. 6 shows yet another embodiment of an optical fiber mountingelement;

FIG. 7 shows still another embodiment of an optical fiber mountingelement;

FIG. 8 shows a further embodiment of an optical fiber mounting element;

FIG. 9 shows one example of an optical fiber microscopy launch systemhaving an example of a fiber insertion portal;

FIG. 10 shows another example of an optical fiber microscopy launchsystem having an example of a fiber insertion portal;

FIG. 11 shows one example of an optical fiber microscopy launch systemconfigured to deliver EMR at an incident angle that is at or above acritical angle for TIR;

FIG. 12 shows one example of an optical fiber microscopy launch systemconfigured to deliver EMR at an incident angle that is less than acritical angle for TIR;

FIG. 13 shows another example of an optical fiber microscopy launchsystem configured to deliver EMR at an incident angle that is less thana critical angle for TIR;

FIG. 14 shows another example of an optical fiber microscopy launchsystem configured to deliver EMR at an incident angle that is at orabove a critical angle for TIR;

FIG. 15 shows one example of an optical fiber microscopy launch systemconfigured to deliver EMR at an incident angle that is at or above acritical angle for TIR and at an incident angle that is less than acritical angle for TIR;

FIG. 16 shows one example of an optical fiber microscopy launch systemconfigured for an example darkfield application;

FIG. 17 shows one example of an optical fiber microscopy launch systemconfigured for an example surface plasmon application;

FIG. 18 shows one example of an optical fiber microscopy launch systemconfigured for an example near-field application;

FIG. 19 shows one example of prism-type TIR microscopy;

FIG. 20 shows one example of through-the-lens TIR microscopy;

FIG. 21 shows one example of an optical fiber microscopy launch system;

FIG. 22 shows another example of an optical fiber microscopy launchsystem;

FIG. 23 shows yet another example of an optical fiber microscopy launchsystem;

FIG. 24 shows still another example of an optical fiber microscopylaunch system;

FIG. 25 shows still yet another example of an optical fiber microscopylaunch system;

FIG. 26 shows one example of an optical fiber microscopy launch systemincluding a sample chamber;

FIG. 27 shows one embodiment of a multiple optical fiber microscopylaunch system;

FIG. 28 shows another embodiment of a multiple optical fiber microscopylaunch system;

FIG. 29 shows one example of a system for modifying a microscope;

FIG. 30 shows another example of a system for modifying a microscope;

FIG. 31 shows yet another example of a system for modifying amicroscope;

FIG. 32 shows still another example of a system for modifying amicroscope; and

FIG. 33 shows one example of an optical fiber microscopy launch system.

DETAILED DESCRIPTION

A system and method is provided that allows the delivery of illuminationand/or excitation radiation to a sample; In one embodiment, an opticalfiber may be positioned proximate a sample slide such that anelectromagnetic radiation (EMR) may be delivered into the sample slide.The EMR that passes into the sample slide illuminates and/or provides anexcitation energy to a sample positioned proximate a surface of thesample slide. In one embodiment, a launch system of the presentdisclosure may be utilized in a variety of microscopy techniques.Example microscopy techniques include, but are not limited to,fluorescence microscopy, TIR microscopy, near-field microscopy, brightfield microscopy, darkfield microscopy, surface plasmon microscopy,particle plasmon microscopy, and any combinations thereof.

FIG. 2 illustrates one embodiment of an optical fiber microscopylaunching system 200. System 200 includes a sample slide 205 having aside 210 and an opposing side 215. Opposing side 215 has a surface 220.Side 210 has a surface 225. Sample slide 205 has a width 230. An opticalfiber 235, which includes a terminal end 240 having a terminal surface245, is positioned proximate side 210 so as to locate terminal end 240to deliver EMR from optical fiber 235 into the substrate 205 to aportion of side 215 at an incident angle 250. In one example, incidentangle 250 may depend on the location of a sample on sample slide 205,the desired microscopy technique, and/or the desired incident angle tothe sample. In an example of TIR microscopy, incident angle 250 is atleast equal to or greater than the critical angle for TIR at surface220. The delivery of EMR may be facilitated in any way that will allowthe EMR to enter substrate 210 such that it is delivered to surface 220at incident angle 250. Structure in substrate 205 or in addition tosubstrate 205 for this facilitation may take place in any location, suchas location 255. In this example, side 210 and opposing side 215 aresubstantially parallel to each other. In another example, illustrated inmore detail below, an optical fiber is positioned proximate a side thatis at an acute angle, such as an about 90 degree angle, to an opposingside.

A sample slide, such as sample slide 205, may include any of a varietyof optically transmissive material. Examples of suitable sample slidematerials include, but are not limited to, glass, fused silica,sapphire, plastic, zirconia, germanium, diamond, and any combinationsthereof. A sample slide may include a material having any index ofrefraction that will allow delivery of EMR from an optical fiber into asample slide and to a region proximate a sample positioned on or nearthe sample slide. In one example, a sample slide includes a silicaglass, with an index of refraction of about 1.40. In another example, asample slide includes a material having an index of refraction higherthan that of its surroundings (e.g., for allowing TIR to occur at aninterface of the substrate and the surrounding). In yet another example,a sample slide may be made of a material having any index of refractionthat will allow TIR at its surface interface with desired samples anddesired external media.

The thickness of a sample slide can vary depending on application. Inone example, a sample slide has a thickness of about 0.125 millimeters(mm) to about 1.0 mm. In another example, a sample slide has two planarsides opposite each other that are large enough to accommodate a desiredsample, each planar side being larger in surface area than a side thatconnects the two planar sides. The two planar sides in this example canbe of any shape, including (but not limited to), square, rectangular,circular, oval, octagonal, triangular, and trapezoidal. In yet anotherexample, a sample slide is a microscope slide.

An optical fiber, such as optical fiber 235, may be an optical EMRwaveguide. Examples of suitable optical fiber materials include, but arenot limited to, single mode, multi mode, polarization maintaining, andany combination thereof. In one example, an optical fiber may beshielded and/or coated as is understood by those of ordinary skill inthe art. An optical fiber may be fabricated with a cladding to enhanceperformance and to isolate the TIR within the fiber from environmentalcoupling effects to the outside surface. Other coatings or armor aresometimes added for mechanical stability. In one example, an opticalaperture at a terminal end (e.g., terminal end 240), can be very smallwith respect to the fiber. For example, a single mode fiber may have adiameter of about 125 microns, while its optical aperture is at about 6microns. In another example, an optical fiber can have an opticalelement at its terminal end, such as at terminal end 240. Examples ofoptical elements that may be present at a terminal end of an opticalfiber include, but are not limited to, a lens, index matching mediumsuch as oil or optical epoxy, optical grating, prism and anycombinations thereof. In one example, a lens is positioned in opticalcommunication with a terminal end of an optical fiber to focus EMRtransmitted therefrom. In this disclosure, discussion of a terminal endof an optical fiber includes the option of one or more optical elementsat that terminal end.

A “proximate position” for an optical fiber with respect to a side of asample slide in the context of the present disclosure is a position thatis close enough to provide EMR from the optical fiber first impinging onthat side and transmitting to the surface of an opposing side of thesample slide at a desired incident angle (e.g., incident angle 250). Inone example, a proximate position is one that will provide EMR to asurface of an opposing side of a sample slide at an angle that willproduce TIR at the surface of the opposing side. The distance from theterminal end of the optical fiber to the sample slide may depend onfactors that include, but are not limited to, the numerical aperture ofthe optical fiber, the index of refraction of the sample slide, thethickness of the sample slide, the angle of the first side of the sampleslide relative to the second side of the sample slide, the index ofrefraction of any material between the terminal end of the optical fiberand the sample slide, and any combinations thereof.

The type of EMR delivered via an optical fiber will depend on thedesired microscopy application the type of sample, and/or the slidematerial. In one example, a suitable EMR for TIR microscopy may be anyEMR that can be delivered using an optical fiber and will cause theexcitation of fluorophores desired to be viewed during any particularTIR microscopy. One of ordinary skill will recognize the variety of EMRsuitable for use with TIR microscopy. Examples of EMR suitable formicroscopy include, but are not limited to, collimated light,non-collimated light, laser light, polarized and non-polarized light,arc lamp light, filament lamp light, flash lamp light, and anycombinations thereof. In one example, a collimated light source isemployed. In another example, a non-collimated light source is employed.In yet another example, EMR may emit from a terminal end of an opticalfiber at more than one angle. In this example, the optical fiber ispositioned so that EMR that is directed to an opposing surface at themost extreme angle is at the desired incident angle (e.g., at least acritical angle for TIR) so that all of the angled light is at least atthe desired angle. One benefit of the present disclosure is that anoptical fiber according to the present disclosure can delivernon-collimated EMR to a sample slide while retaining an illuminationarea of defined geometry that can be easily adjusted to accommodateexperimental variations. Previous systems, such as in TIR microscopysystems, used collimated EMR, usually a laser, due to the need to directthe EMR to an objective lens or prism using the various opticalequipment discussed above, such as beam expanders, mirrors, and focusinglenses.

In one example wherein the material between a terminal end of an opticalfiber and a side of a sample slide (e.g., having an index of refractionof about 1.5) is air (index of refraction of about 1) or water (index ofrefraction of about 1.33), the sample slide may require a modificationto the side upon which EMR from the terminal end of the optical fiberfirst impinges in order to allow a portion of the EMR to be transmittedto a surface of an opposing side of the sample slide. Examples of suchmodifications are discussed below and include, but are not limited to,providing a bevel in the side of the sample slide, the bevel having asurface that is at an angle with respect to the terminal end of anoptical fiber to allow transmission of EMR into the sample slide;providing a micro grating, a prism, a mounting element, a couplingmedium, a fresnel lens; and any combinations thereof.

An optical fiber can be held in position relative to a side of a sampleslide in any way that will maintain the desired angle of incidence ofthe EMR at a surface of an opposing side. In one example, a mountingelement, as discussed below, is used to hold an optical fiber inposition. In yet another example, an optical fiber is positioned in afiber insertion portal, discussed below, that is in the sample slide oran additional mounting element.

In one embodiment, the facilitation of the delivery of EMR into a sampleslide, such as sample slide 205, may include an optical fiber mountingelement as part of the sample slide and/or optically coupled thereto.FIG. 3 illustrates one example of a sample slide 305 having a first side310 and a second side 315. Second side 315 has a surface 320. An opticalfiber 335 is positioned proximate first side 310 utilizing an opticalfiber mounting element 355 optically coupled to first side 310 of sampleslide 305.

An optical fiber mounting element may be configured in a way and/or mayinclude any material that will allow EMR to pass from a terminal end ofan optical fiber (e.g., terminal end 340 of optical fiber 335) to asample slide, while positioning the optical fiber to deliver EMR to asurface of a second side (e.g. side 315) of the sample slide at adesired angle of incidence, such as angle 350. Example mounting elementmaterials include, but are not limited to, prism, glass, plastic,sapphire, metal, an optical coupling medium, and any combinationsthereof. An optical coupling medium may be any material that facilitatestransmission of EMR from one material to another (e.g., from an opticalfiber mounting element to a sample slide, from an optical fiber to anoptical fiber mounting element). Examples of an optical coupling mediuminclude, but are not limited to, water, an optical adhesive, glycerol,an optical oil, an optical grating (e.g., a micrograting), a prism, abeveled window, a lens, and any combination thereof. In one example, anoptical coupling medium may be utilized to bring optical fiber 335 intooptical communication with optical fiber mounting element 355. Inanother example, a coupling medium may be utilized to connect opticalfiber mounting element 355 in optical communication with sample slide305.

An optical fiber mounting element may have a substantially similar indexof refraction as a sample slide or a different index of refraction as asample slide as long as an optical fiber is positioned so to deliver EMRat a desired angle of incidence. In one example, the index of refractionof an optical fiber mounting element is the same, or substantially thesame, as a sample slide. In such an example, EMR from the terminal endof an optical fiber in optical communication with an optical fibermounting element will enter the optical fiber mounting element at afirst angle. Since the index of refraction of the optical fiber mountingelement, which is in optical communication with the sample slide, is thesame as the index of refraction of the sample slide, the EMR does notrefract, or refracts only minimally, at the interface between theoptical fiber mounting element and the sample slide. In another example,an optical fiber mounting element has an index of refraction thatdiffers from the index of refraction of a sample slide to which it is inoptical communication. In such an example, EMR from the terminal end ofan optical fiber in optical communication with the optical fibermounting element will enter the optical fiber mounting element at afirst angle. Since the index of refraction of the optical fiber mountingelement is different than the index of refraction of the sample slide,the EMR refracts at the interface of the optical fiber mounting elementand the sample slide. Thus, the angle of incidence at an opposingsurface of the sample slide is different than the first angle. Oneadvantage to such an example is that an optical fiber can be positionedwith respect to a sample slide at an angle that would not otherwiseprovide light to the internal surface of the second side of a sampleslide at a desired angle of incidence (e.g., a critical angle for TIR).Due to the refraction at the interface between the optical fibermounting element and the sample slide, a critical angle can be attainednonetheless. This can be useful when microscope equipment or otherimpediments make it difficult to position the optical fiber itself at anangle that would otherwise provide EMR at the desired angle of incidenceat the opposing surface.

An optical fiber mounting element, such as optical fiber mountingelement 355 can be any shape suitable for properly positioning anoptical fiber with respect to a sample slide. Example shapes of anoptical fiber mounting element include, but are not limited to, round,triangular, square, rectanglular, hemispherical, trapezoidal, andparallelogram. In one example, an optical fiber mounting element mayinclude a fiber insertion portal. In another example, an optical fibermounting element may be a fiber insertion portal directly in a side of asample slide. In one example, optical fiber mounting element 355 isconfigured such that at least some of the EMR passing into the sampleslide and to the opposing side has an angle of incidence that is at orabove a critical angle for TIR to occur at the surface of the opposingside. In another example, optical fiber mounting element 355 isconfigured such that at least some of the EMR passing into the sampleslide and to the opposing side has an angle of incidence that is below acritical angle for TIR and passes through the opposing side to a samplepositioned proximate the opposing side and/or to a region surroundingthe sample slide.

In one example, an optical fiber mounting element may be of an opticallyopaque material which holds, contains, or locates an optical couplingmedium. In another example, an optical fiber mounting element maycontain reflective or refractive optics or optical properties. Anoptical fiber mounting element maybe a monolithic material with a sampleslide. In another example, an optical fiber mounting element may be aseparate material (with similar or different index of refraction) thatis, or can be, positioned in optical communication with a side of asample slide. In another example, an optical fiber mounting element is aseparate material which is optically opaque, and provides a portal forfiber insertion into an optical coupling medium which facilitates EMRtransmission into the sample slide through a window or optical portal.In still another example, an optical fiber mounting element may includerefractive elements such as a lens to bend the light rays prior totransmission into the sample slide. In another example, an optical fibermounting element simply holds the fiber at a suitable angle proximate toa grating or beveled edge where the EMR enters the sample slide.

An optical fiber mounting element may be configured to allow an opticalfiber to move from one position to another to allow for adjustment inthe angle of incidence at an opposing side. Such movement may allowmicroscopy with one technique (e.g., TIR) and ease of movement to anadditional technique (e.g., darkfield).

FIG. 4A illustrates a cross-sectional view of one example of an opticalfiber mounting element 455. Optical fiber mounting element 455 includesa fiber insertion portal 460 having a fiber insertion axis 462. Althoughfiber insertion portal 460 is shown in FIG. 4A as being in a top side ofan optical fiber mounting element, it is contemplated that a fiberinsertion portal can be in another side of an optical fiber mountingelement. Fiber insertion portal 460 is an appropriate size and shape toreceive an optical fiber. In one example, a fiber insertion portal, suchas fiber insertion portal 460, is conformally configured to receive anoptical fiber. In another example, any space in fiber insertion portal460 that would remain after insertion of an optical fiber can be filledwith an optical coupling medium. Fiber insertion portal 460 includes anend 464, a side 466, and a side 468. End 464 can be of any shape and atany angle to sides 466 and 468. In one example, end 464 is configured atsuch an angle with respect to EMR from an optical fiber inserted thereinso to allow transmission of at least a portion of the EMR into theoptical fiber mounting element, for example such an angle that is aboutnormal to the EMR. End 464 is shown as being at a right angle to sides466 and 468. In one example, end 464 is at an angle to sides 466 and 468so to conformally receive an optical fiber having an angled terminalend. In another example (not shown), end 464 is shaped to conformallyreceive an optical fiber having a terminal end including an opticalelement (e.g., a lens, prism, grating). One of ordinary skill willrecognize a variety of combinations of shapes of a fiber insertionportal and an optical fiber that are available, including with an optionof filling extra space with a coupling medium.

Optical fiber mounting element 455 has a contact side 470 for contactingwith a side of a sample slide. FIG. 4B shows a plan view of opticalfiber mounting element 455 with fiber insertion portal 460. Opticalfiber mounting element 455 is shown as a rectangular block. As discussedabove, an optical fiber mounting element can be any shape suitable forproperly positioning an optical fiber with respect to a sample slide.Optical fiber mounting element 455 shows one fiber insertion portal 460.It is contemplated that a plurality of fiber insertion portals may beprovided in one optical fiber mounting element. This may facilitatemultiple fiber excitation and/or illumination as is discussed furtherbelow.

FIG. 5A illustrates a cross-sectional view of one example of an opticalfiber mounting element 555. Optical fiber mounting element 555 includesa fiber insertion portal 560 having a fiber insertion axis 562. Fiberinsertion portal 560 is an appropriate size and shape to receive anoptical fiber. Fiber insertion portal 560 includes an end 564, a side566, and a side 568. End 564 is open at a contact side 570. Contact side570 is configured to contact with a side of a sample slide. In oneexample, a terminal end of an optical fiber inserted in fiber insertionportal 560 may extend to end 564. An optical coupling medium (e.g., anoil) may be utilized in fiber insertion portal 560 to provide opticalcoupling between the terminal end and a sample slide proximate tosurface 570. Optical fiber mounting element 555 may be constructed froma variety of materials. In one example optical fiber mounting element555 may be constructed of an optically opaque material. FIG. 5Billustrates a plan view of optical fiber mounting element 555 includingfiber insertion portal 560.

A fiber insertion portal can be at any appropriate depth for properlypositioning an optical fiber. When an optical fiber mounting element isused in conjunction with a mechanical coupling or other coupling, thedepth of a fiber insertion portal may not be required to support anoptical fiber itself. FIG. 6A illustrates a cross-sectional view of anexample of an optical fiber mounting element 655 having a fiberinsertion portal 660. Fiber insertion portal 660 is shaped as a bevel ina side 674 of optical fiber mounting element 655. FIG. 6B illustrates aplan view of optical fiber mounting element 655 and fiber insertionportal 660. Fiber insertion portal 660 is shown here as a bevel acrossthe surface of side 674.

FIG. 7 A illustrates a cross-sectional view of an example of an opticalfiber mounting element 755 having a grating 760 in side 774. FIG. 7Billustrates a plan view of optical fiber mounting element 755 andgrating 760. As one of ordinary skill will recognize, a beveled fiberinsertion portal or a grating in the surface need not extend the entiresurface of side. Further, in another example, a beveled fiber insertionportal or a grating may be made directly in a side of a substrate.

Some variations of the fiber insertion portal provide optical couplingfacility only. In FIG. 6 and FIG. 7, EMR is transmitted into the elementbecause of the angle of the bevel or grating. These examples may requireadditional mechanical facility for holding the optical fiber such thatEMR is directed from a terminal end of the optical fiber to fiberinsertion portal 660, grating 760 such that the EMR will enter theoptical fiber mounting element and transmit into a sample slideproximate to a surface of the optical fiber mounting element.

In another embodiment, not shown, an optical fiber mounting element,such as optical fiber mounting element 455, can have an optical fiberinserted in its fiber insertion portal. The optical fiber may in somecases be made to be in optical communication with the optical fibermounting element with the use of an optical coupling medium or someother technique. In one example, the optical fiber can be attached tothe optical fiber mounting element using an optical adhesive as theoptical coupling medium or some other form of adhering technique. Inanother example, the end of the optical fiber not coupled with theoptical fiber mounting element can be terminated with any opticalcoupling known to those of ordinary skill and/or may be connected to asource of EMR. In still another example, a system of the presentdisclosure can be used to modify a sample slide, as discussed furtherbelow.

FIG. 8 illustrates yet another embodiment of an optical fiber mountingelement 805. Optical fiber mounting element 805 can have a fiberinsertion portal 860 that is filled with an optical coupling medium 865,such as an optical adhesive, glycerol, an optical oil, and/or othercoupling medium. In one example, the opening of the fiber insertionportal can be sealed with an appropriate sealing element 875. Examplesof an appropriate sealing element include, but are not limited to, anadhesive tape, a plug, silicone rubber, thermoplastic, epoxy resin,clay, putty, and any combinations thereof. Prior to use of optical fibermounting element 855, sealing element 875 may be removed and an opticalfiber inserted into fiber insertion portal 860. Such an optical fibermounting element may be provided alone or pre-attached/optically coupledto a sample slide.

FIG. 9 illustrates an example of a sample slide 905 having a side 910 inoptical communication with optical fiber mounting element 955. Opticalfiber mounting element 955 includes a fiber insertion portal 960 havinga fiber insertion axis 962. Optical communication can be facilitatedusing an optical coupling medium, such as an optical adhesive, glycerol,an optical oil, and/or other coupling medium.

FIG. 10 illustrates yet another embodiment of a sample slide 1005 havinga side 1010 with a surface 1025, and a side 1015 with a surface 1020.Sample slide 1110 includes a optical fiber mounting element that is afiber insertion portal 1060 having a fiber insertion axis 1062.

In another example, not shown, a sample slide, such as sample slide1005, can have an optical fiber inserted in fiber insertion portal 1060.The optical fiber may in some cases be made to be in opticalcommunication with the sample slide with the use of an optical couplingmedium or some other technique. The optical fiber can be attached to thesample slide using an optical adhesive as an optical coupling medium orsome other form of adhering technique. In one example, the end of theoptical fiber not coupled with the sample slide can be terminated withany optical coupling known to those of ordinary skill or may beconnected directly to a source of EMR.

In yet another example, not shown, a sample slide may have a fiberinsertion portal filled with an optical coupling medium with a sealingelement, as was discussed above with respect to a fiber insertion portalof an optical fiber mounting element above.

As was discussed above, an optical fiber microscopy launch system and/ormethod of the present disclosure may be utilized in a variety ofmicroscopy applications.

FIG. 11 illustrates one example optical fiber microscopy launch system1100. System 1100 includes a sample slide 1105 having a first side 1110and a second opposing side 1115. Second side 1115 has a surface 1120 andfirst side 1110 has a surface 1125. Optical fiber 1135 has a terminalend 1140 that is in proximity to first side 1110. Optical fiber 1135 isoptically coupled with an optical fiber mounting element 1155 that isoptically coupled to surface 1125. Optical fiber 1135 is positioned byoptical fiber mounting element 1155 such that EMR from terminal end 1140is directed through an interface between optical fiber mounting element1155 and sample slide 1105 to surface 1120 at an incident angle 1150that is at least a critical angle for TIR. The EMR totally internallyreflects at surface 1120 and continues TIR through sample slide 1105.TIR fluorescent microscopy (TIRFM) may be implemented by observingfluorescence of a sample on a surface of sample slide 1105 at a point ofTIR.

Terminal end 140 of optical fiber 1135 is shown as being in conformalcontact with respect to a surface of optical fiber mounting element1155. In an alternative embodiment, a terminal end of an optical fiber,such as terminal end 140, may be in contact with a surface of an opticalfiber mounting element, such as optical fiber mounting element 1155,such that it does not conform and provides a refraction of EMR as itpasses from the terminal end into an optical fiber mounting element1155. In one example, terminal end 140 may be configured at an anglewith respect to optical fiber 1135 (i.e., at a non-normal angle). Inanother example, a lens or other refracting material may be utilizedbetween a terminal end of an optical fiber and an optical fiber mountingelement. In yet another example, one or more bevels in the surface of anoptical fiber mounting element may provide a desired refraction. Instill another example, a coupling medium with a mismatched index ofrefraction may be provided between a terminal end and an optical fibermounting element to provide a desired refraction of EMR. One benefitthat may be provided by refracting an EMR as it passes from a terminalend of an optical fiber (e.g., terminal end 1140) to an optical fibermounting element (e.g., optical fiber mounting element 1155) includesthe ability to provide flexibility in an incident angle (e.g., incidentangle 1150).

Optical fiber 1135 is shown for convenience of illustration in FIG. 11as having space between optical fiber 1135 and the walls of a fiberinsertion portal of optical fiber mounting element 1155. An opticalfiber, such as optical fiber 1135, may be positioned in a fiberinsertion portal of an optical fiber mounting element such that there issubstantially no space between the side of the optical fiber and theoptical fiber mounting element.

FIG. 12 illustrates another example optical fiber microscopy launchsystem 1200. System 1200 includes a sample slide 1205 having a firstside 1210 and a second opposing side 1215. Second side 1215 has asurface 1220 and first side 1210 has a surface 1225. Optical fiber 1235has a terminal end 1240 that is in proximity to first side 1210. Opticalfiber 1235 is optically coupled with an optical fiber mounting element1255 that is optically coupled to surface 1225. Optical fiber 1235 ispositioned by optical fiber mounting element 1255 such that EMR fromterminal end 1240 is directed through an interface between optical fibermounting element 1255 and sample slide 1205 to surface 1220 at anincident angle 1250 that is less than a critical angle for TIR. The EMRtotally transmits through surface 1220 and refracts.

FIG. 13 illustrates yet another example optical fiber microscopy launchsystem 1300. System 1300 includes a sample slide 1305 having a firstside 1310 and a second opposing side 1315. Second side 1315 has asurface 1320 and first side 1310 has a surface 1325. Optical fiber 1335has a terminal end 1340 that is in proximity to first side 1310. Opticalfiber 1335 is optically coupled with an optical fiber mounting element1355 that is optically coupled to surface 1325. Optical fiber 1335 ispositioned by optical fiber mounting element 1355 such that EMR fromterminal end 1340 is directed through an interface between optical fibermounting element 1355 and sample slide 1305 to surface 1320 at anincident angle 1350 that is just barely less than a critical angle forTIR. The EMR totally transmits through surface 1320 and refracts nearlyperpendicular to surface 1320.

FIG. 14 illustrates still another example optical fiber microscopylaunch system 1400. System 1400 includes a sample slide 1405 having afirst side 1410 and a second opposing side 1415. Second side 1415 has asurface 1420 and first side 1410 has a surface 1425. Optical fiber 1435has a terminal end 1440 that is in proximity to first side 1410. Opticalfiber 1435 is optically coupled with an optical fiber mounting element1455 that is optically coupled to surface 1425. Optical fiber 1435 ispositioned by optical fiber mounting element 1455 such that EMR fromterminal end 1440 is directed through an interface between optical fibermounting element 1455 and sample slide 1405 to surface 1420 at anincident angle 1450 that is just barely greater than a critical anglefor TIR. The EMR totally internally reflects at surface 1420 at a lowangle and transmits through sample slide 1405. In this example, a samplefor TIRFM would need to be positioned at the single point of TIR onsurface 1420.

In practice, total collimation of EMR emerging from a terminal end of anoptical fiber is not always achieved and/or desired. In one example,divergent EMR may provide a narrow band of angles of EMR with some ofthe EMR directed to an opposing surface at or above a critical angle forTIR and some of the EMR directed to an opposing surface at an angle lessthan a critical angle for TIR. FIG. 15 illustrates a further exampleoptical fiber microscopy launch system 1500. System 1500 includes asample slide 1505 having a first side 1510 and a second opposing side1515. Second side 1515 has a surface 1520 and first side 1510 has asurface 1525. Optical fiber 1535 has a terminal end 1540 that is inproximity to first side 1510. Optical fiber 1535 is optically coupledwith an optical fiber mounting element 1555 that is optically coupled tosurface 1525. Optical fiber 1535 is positioned by optical fiber mountingelement 1555 such that EMR from terminal end 1540 is directed through aninterface between optical fiber mounting element 1555 and sample slide1505 to surface 1520 at an various incident angles 1550, some of whichare just barely at or greater than a critical angle for TIR and othersthat are just barely below a critical angle for TIR. Some of the EMRtotally transmits at a low angle to surface 1520 and some of the EMRtotally internally reflects at a low angle to surface 1520. Some EMR istraveling substantially parallel to surface 1520.

In one example, an optical fiber may be positioned utilizing an opticalfiber mounting element such that its position may be varied in order totake advantage of the differing reflection and transmissioncharacteristics at different incident angles. In one example, an EMRthat transmits through an opposing surface (e.g., surface 1520) andpropagates nearly parallel to that surface may provide illumination of asample on that surface. In another example, an EMR that totallyinternally reflects may provide an evanescent field, as discussedfurther below, that is used as an excitation energy for TIRFM. In yetanother example, a combination of illumination and excitation may alsobe provided.

FIG. 16 illustrates one example darkfield microscopy application 1600utilizing an incident angle of less than a critical angle for TIR.Application 1600 includes a sample slide 1605 having a first side 1610and a second opposing side 1615. Second side 1615 has a surface 1620 andfirst side 1610 has a surface 1625. Optical fiber 1635 has a terminalend 1640 that is in proximity to first side 1610. Optical fiber 1635 isoptically coupled with an optical fiber mounting element 1655 that isoptically coupled to surface 1625. Optical fiber 1635 is positioned byoptical fiber mounting element 1655 such that EMR from terminal end 1640is directed through an interface between optical fiber mounting element1655 and sample slide 1605 to surface 1620 at an incident angle 1650that is less than a critical angle for TIR. The EMR totally transmitsthrough surface 1620 and refracts at an angle 1667. A sample 1670 ispositioned at surface 1620 such that it is illuminated by the refractedEMR. The EMR continues at about angle 1667 such that it does not enteran objective lens 1695. Such an example may be useful for a darkfieldmicroscopy applications. In one example, sample slide 1605 may bepositioned with respect to objective lens 1695 so that objective lens1695 may detect scattered and/or fluorescent energy (e.g., light) fromsample 1670 as the EMR illuminates sample 1670.

FIG. 17 illustrates one example surface plasmon microscopy application1700. Application 1700 includes a sample slide 1705 having a first side1710 and a second opposing side 1715. Second side 1715 has a surface1720 and first side 1710 has a surface 1725. Optical fiber 1735 has aterminal end 1740 that is in proximity to first side 1710. Optical fiber1735 is optically coupled with an optical fiber mounting element 1755that is optically coupled to surface 1725. Optical fiber 1735 ispositioned by optical fiber mounting element 1755 such that EMR fromterminal end 1740 is directed through an interface between optical fibermounting element 1755 and sample slide 1705 to surface 1720 at anincident angle 1750. In one example, incident angle 1750 may be as lowas zero degree (i.e., perpendicular to surface 1725). A surface plasmonreflective layer 1765 is positioned at the point of incidence of the EMRand a sample 1770 is positioned in contact with surface plasmonreflective layer 1765 opposite of sample slide 1705. Example surfaceplasmon reflective layers include, but are not limited to, a thin filmof a material such as gold, a silver, copper, aluminium, sodium, indium,and any combinations thereof. When incident angle 1750 allowstransmission of the EMR through surface 1720, the EMR reflects fromlayer 1765, which induces an evanescent field-like non-radiativetransfer of energy to sample molecules in close proximity to layer 1765.Such an example may be useful for fluorescence microscopy, measurementof a density change in sample 1770, measurement of a surface bindingenergy, fluorescence-based immunoassays, and any combinations thereof.In another example, a surface plasmon may be applied to multiplesurfaces of a sample slide. In one aspect application 1700 may provide asimplified process and equipment for conducting a surface plasmontechnique. In one example, an objective lens 1795 (or other detectionelement) may be positioned to view and/or detect an energy from sample1770.

FIG. 18 illustrates one example of a near-field microscopy application1800. Application 1800 includes a sample slide 1805 having a first side1810 and a second opposing side 1815. Second side 1815 has a surface1820 and first side 1810 has a surface 1825. Optical fiber 1835 has aterminal end 1840 that is in proximity to first side 1810. Optical fiber1835 is optically coupled with an optical fiber mounting element 1855that is optically coupled to surface 1825. Optical fiber 1835 ispositioned by optical fiber mounting element 1855 such that EMR fromterminal end 1840 is directed through an interface between optical fibermounting element 1855 and sample slide 1805 to surface 1820 at anincident angle 1850. In one example, incident angle 1850 may be as lowas zero degree (i.e., perpendicular to surface 1825). A coating 1865 orother element that provides reflection for the EMR and one or more holesand/or slits 1867 in the coating that have a width that is smaller thanthe wavelength of the EMR. The EMR at one or more holes and/or slits1867 acts as a point source which produces an evanescent field thatpropagates into sample 1870. Such an example may be useful forfluorescence microscopy and other techniques. In one example, anobjective lens 1895 (or other detection element) may be positioned toview and/or detect an energy from sample 1870. Although, FIG. 18illustrates one or more holes and/or slits 1867 as a single hole/slitfor exemplary purposes, a plurality of holes/slits may be used toprovide a plurality of near-field effects in one or more samples. In oneexample, a single optical fiber, such as optical fiber 1835, may beemployed to illuminate a plurality of holes/slits to provide anear-field effect. In an alternative embodiment, one or more holesand/or slits 1867 may have a width that is larger than the wavelength ofthe EMR in order to provide an evanescent field for TIRFM application.

In another embodiment, an optical fiber microscopy launch system andmethod may be utilized with TIR microscopy (e.g., totally internallyreflected fluorescence microscopy, TIRFM). Features of an exampleoptical fiber microscopy launch system and method will be described withrespect to TIRFM below. Those skilled in the art will recognize fromthis disclosure that certain features may also apply to non-TIRmicroscopy utilizing an optical fiber microscopy launch system accordingto the present disclosure.

Referring again to FIG. 1, in TIRFM, when EMR is reflected off of asample slide/sample interface, such as interface 110, at or above thecritical angle, an evanescent electromagnetic field, such as evanescentfield 170, is formed at the interface and decays exponentially into thesample. This field can be used as a fluorescence excitation source. Oneadvantage of this excitation is that it has an extremely well definedarea of excitation and shallow depth, resulting in only thosefluorophores within the evanescent field being excited. This candramatically reduce background fluorescence and improve signal to noiseand spatial resolution.

In prior art systems and procedures, TIRFM is expensive. Part of theexpense is related to the cost of complicated equipment needed to builda TIRFM microscope or to convert a non-TIRFM microscope to performTIRFM. Two examples of TIRFM techniques include “prism-type” TIRFM and“through the lens” TIRFM. FIG. 19 illustrates one example of aprism-type TIRFM setup 1900. A sample 1910 is affixed to a sample slide1920. A prism 1930 with appropriate optical properties, such as index ofrefraction, to match the optical properties of sample slide 1920 ispositioned on the sample slide. Laser light 1940 from a source 1950 isreflected using optical equipment 1960 through focusing lens 1970 andinto prism 1930. In this example, optical equipment 1960 is shown as asingle mirror for simplicity. However, typically optical equipment 1960would include various optical components needed to direct laser light1940 from source 1950 at an appropriate angle to a microscope stage andinto prism 1930. Prism 1930 directs laser light 1940 at an interface1980 between sample 1910 and sample slide 1920 at or above the criticalangle so as to induce TIR. One of the drawbacks to such a system is thatit is difficult or impossible with typical microscopes to positionoptical equipment 1960 such that it translates with the microscope stageduring focusing of the microscope. Changes in the position of the sampleslide during focusing without corresponding changes in optical equipment1960 and/or focusing lens 1970 can result in a negative impact on TIR,including loss of critical angle and/or alignment to the field of view.

FIG. 20 illustrates one example of “through the lens TIRFM.” IncomingEMR 2010 passes through a focusing lens 2020 and is directed by areflective element 2030 to an objective lens 2040. Incoming EMR 2010 isincident at an interface 2050 of a first material 2060 and a secondmaterial 2070. Total internally reflected energy 2080 is reflected backthrough objective lens 2040 and directed by reflective element 2030through focusing lens 2020. Reflective element 2030 is selected to allowincoming EMR 2010 and TIR energy 2080 to reflect, while allowing desiredviewing energy (such as fluorescence energy) to pass to an observationelement 2090, such as a fluorescence detector or camera. Through thelens TIRFM requires high numerical aperture (NA) objective lenses toachieve TIR. In one example, an objective lens having an NA of 1.35 ormore is required. In another example, an objective lens having an NA of1.45 or more is required. One example where a system would benefit froman objective lens having an NA of 1.45 or more would be a glass/waterinterface.

FIG. 21 illustrates one example of a TIR system 2100 according to thepresent disclosure. TIR system 2100 includes a sample slide 2110 havinga side 2120 and an opposing side 2130. Opposing side 2130 has a surface2136. Side 2120 has a surface 2126. Sample slide 2110 has a width 2112.An optical fiber 2140, which includes a terminal end 2144 having aterminal surface 2146, is positioned proximate side 2120 so as to locatethe terminal end to deliver EMR from the optical fiber into the sampleslide 2110 to side 2130 at an incident angle 2150. Incident angle 2150is at least equal to or greater than the critical angle for TIR atsurface 2136. The delivery of EMR may be facilitated in any way thatwill allow the EMR to enter sample slide 2110 such that it is deliveredto surface 2136 at or above a critical angle for TIR. Structure insample slide 2110 or in addition to sample slide 2110 for thisfacilitation may take place in any location, such as location 2147. Inthis example, side 2120 and opposing side 2130 are substantiallyparallel to each other. In another example, illustrated in more detailbelow, an optical fiber is positioned proximate a side that is at anacute angle, such as an about 90 degree angle, to an opposing side.Examples of ways to facilitate delivery of EMR at or above a criticalangle include those described herein and include, but are not limitedto, use of mounting element, use of an optical coupling medium, and anycombinations thereof.

FIG. 22 illustrates one example of a sample slide 2210 having a side2220 and a side 2230. Optical fiber 2240 is held in position withrespect to sample slide 2210 by one example of a mechanical coupling,i.e., mechanical coupling 2245, such that a critical angle 2250 ismaintained. In this example one or more ways of facilitating delivery ofEMR at or above a critical angle may be included in at least location2247. Other examples of a mechanical coupling include, but are notlimited to, thermoplastic molded assembly, optical adhesive, machinedmetal or plastic, and any combinations thereof.

FIG. 23 illustrates one example of a sample slide 2310 having a side2320 (with surface 2326) and a side 2330 (with surface 2336). A terminalend 2334 of an optical fiber 2340 is positioned within an opticalcoupling medium 2360 (i.e., an optical fiber mounting element), which isin optical communication with sample slide 2310, such that a criticalangle 2350 is maintained. In one example, a coupling medium does notinterfere with continued TIR in a sample slide. EMR 2344 is incident atsurface 2336 at critical angle 2350. EMR 2344 totally internallyreflects at a point 2338. The first reflection of EMR 2344 on surface2326 occurs at a point 2328. In FIG. 23, an edge 2362 of coupling medium2360 is spaced from point 2338. If optical coupling medium 2360 were toextend over point 2328, EMR 2344 would pass through an interface betweencoupling medium 2360 and sample slide 2310, due to their respectiveindices of refraction. However, in an example such as that in FIG. 23,critical angle 2350 is chosen such that TIR occurs at the interfacebetween sample slide 2310 and medium 2390, between sample slide 2310 anda medium 2392, and between sample slide 2310 and a sample 2370. In thisexample, sample 2370 is shown for illustrative purposes in opticalcommunication with surface 2336 of second side 2330. One of ordinaryskill will recognize that in use of sample slide 2310, sample 2370 maybe at a different location, such as in optical communication withsurface 2326 of first side 2320. In another example (not shown), theedge of a coupling medium may extend past the point at a surface, suchas surface 2326, where EMR that has totally internally reflected from asurface of an opposing side impinges. In such an example, as mentionedabove, the EMR would exit the sample slide at that point. TIR microscopyis still possible if the sample is positioned at the point of TIR at thesurface of the opposing side. The sample will be subjected to theevanescent field produced by the TIR and the resultant fluorescence ofany fluorophores can be observed.

FIG. 24 illustrates an example optical fiber mounting element 2480.Optical fiber mounting element 2480 includes a fiber insertion portal2490 having a fiber insertion axis 2492. A side 2482 of optical fibermounting element 2480 is in optical communication with a side 2420 of asample slide 2410. Fiber insertion portal 2490 has an optical fiber 2440therein at a position such that EMR from a terminal end 2444 of theoptical fiber is incident at a surface 2436 of a second side 2430 ofsample slide 2410 at an angle 2450 that is at least equal to or greaterthan a critical angle for TIR.

FIG. 25 illustrates another example of a TIR system 2500. A sample slide2510 includes a side 2520, a side 2530, and a side 2538. Side 2530includes a surface 2536. Side 2538 is at an acute angle to side 2530. Anoptical fiber 2540, having a terminal end 2544, is positioned proximateside 2538 of sample slide 2510 so to deliver EMR from terminal end 2544to surface 2536 of side 2530 at an incident angle 2550. Incident angle2550 is at least a critical angle for TIR. Optical fiber 2540 can bepositioned with any of the ways discussed herein including, but notlimited to, mechanical coupling, mounting element (e.g., opticalcoupling medium, fiber insertion portal, etc.), and any combinationsthereof. As is also clear from this disclosure, optical fiber 2540alternatively could have been positioned proximate other sides with adifferent opposing side at which TIR would be initiated. In one example,optical fiber 2540 could have been positioned proximate side 2530 withside 2520 being the opposing side at which TIR would be initiated. In ananother example, optical fiber 2540 positioned proximate side 2538 couldbe positioned so to deliver EMR to surface 2526 at an angle that is atleast a critical angle for TIR. It is contemplated that the variousalternative placements of an optical fiber equally apply to the otherexamples and embodiments disclosed herein.

A sample slide may be part of a sample chamber. One of ordinary skillwill be familiar with a variety of sample chambers. One example of asample chamber having a sample slide according to the present disclosureis illustrated in FIG. 26. A sample chamber 2605 includes a sample slide2610. Sample chamber 2605 includes sample slide 2610 as a top portionand an enclosing element 2615, such as a cover slip, as a lower portion.A spacer 2618 is positioned between sample slide 2610 and enclosingelement 2615 to form a chamber area 2625. Sample slide 2610 includes aside 2620 and an opposing side 2630 with a surface 2636. An opticalfiber 2640 is positioned proximate side 2620 so as to position opticalfiber 2640 to deliver an EMR from its terminal end 2644 having aterminal surface 2646 into sample slide 2610 by one or more ways offacilitating delivery of EMR located at least partially within location2647 at an angle 2650 that is at least a critical angle for TIR atsurface 2636. Optical fiber 2640 can be positioned in any of the waysdiscussed in this disclosure. One non-limiting example of such a waywould be using a mounting element with a fiber insertion portal asdiscussed above. Although optical fiber 2640 is positioned in FIG. 26proximate side 2620, as previously discussed, optical fiber 2640 canalternatively be positioned proximate another side of sample slide 2610.In the example of FIG. 26, a sample 2670 is positioned at surface 2636.When EMR is delivered to surface 2636 at or above a critical angle forTIR, the EMR totally internally reflects causing an evanescent wave thatemanates from surface 2636 into sample 2670 causing any fluorophorespresent in the evanescent wave to fluoresce. As is the case with any ofthe embodiments and examples described herein, fluorescence of a samplecan be detected by a fluorescence detection element, such asfluorescence detection element 2697 in FIG. 26. Fluorescence detectionelement 2697 is positioned to view fluorescence via microscope optics2695.

Examples of fluorescence detection elements include, but are not limitedto, film; video camera, such as an intensity-enhanced video camera;charged coupled diode (CCD), such as a cooled scientific CCD; avalanchephotodiode; photomultiplier; time-lapse cinemicrography; siliconphotodiode; and any combinations thereof.

In a further embodiment, multiple optical fibers may be utilized in anoptical fiber launching system and/or method of the present disclosure.The multiple optical fibers may be brought into proximity to a side of asample slide in a variety of ways as described herein with reference tothe single optical fiber examples. Examples of how to bring multiplefibers into proximity to a side of a sample slide according to thepresent disclosure include, but are not limited to, having multipleoptical fibers in optical communication with a single optical fibermounting element, having each optical fiber in optical communicationwith a different optical fiber mounting element, and any combinationsthereof. The multiple optical fibers may be arranged in a variety ofways to achieve differing results with respect to directing EMR througha sample slide to an opposing side of the sample slide (e.g., for TIR,transmission to a sample, and a combination thereof). Examples ofarrangements of multiple fibers include, but are not limited to, havingterminal ends directing EMR perpendicular to each other, having terminalends directing EMR parallel to each other, having terminal endsdirecting EMR such that the EMR from each optical fiber overlaps, havingterminal ends directing EMR such that the EMR is directed at differentportions of an opposing side of a sample slide, and any combinationsthereof.

Example applications for multiple optical fibers include, but are notlimited to, illuminating and/or providing excitation energy to differentregions of a single sample in proximity to a sample slide, illuminatingand/or providing excitation energy to multiple samples in proximity to asample slide, providing EMR having multiple polarization states,providing EMR having multiple wavelengths to a single location,providing EMR having multiple wavelengths to multiple locations, and anycombinations thereof. In one example, multiple optical fibers may beprovided that are aligned substantially parallel to each other with atleast one optical fiber providing an EMR through a sample slide to anopposing side such that the EMR is polarized perpendicular to a surfaceof the opposing side and at least one other optical fiber providing anEMR through the sample slide to the opposing side such that the EMR ispolarized parallel to the surface of the opposing side. Such anorthogonal arrangement of polarization may also be obtained positioningthe multiple fibers substantially perpendicular to each other. Thoseskilled in the art will recognize numerous applications (e.g., TIRapplications) that require known and/or controlled polarization and/orwavelength. Applications include, but are not limited to providingexcitation energy to single fluorescent molecules and quantum dots,providing excitation energy to fluorescent molecules that requireexcitation at different wavelengths, triggering a photo activatedcompound (e.g., blebbistatin) by specific wavelengths, and anycombinations thereof. In an alternative embodiment, multiple wavelengthsof EMR may be delivered via a single optical fiber. In one example, anoptical fiber having multiple wavelength capacity as is known to thoseof ordinary skill may be utilized with a wavelength selecting element(e.g., a dichotic mirror, one or more shutters, multiple lasers, anAcouso-Optic-Tunable-Filter (AOTF), or any combination thereof) toselect the desired multiple wavelengths entering the optical fiber. Inanother example, multiple wavelengths maybe alternated on an opticalfiber. In yet another example, multiple wavelengths may besimultaneously transmitted on an optical fiber. In still anotherexample, an intensity of a wavelength on an optical fiber may bemodulated.

In one example, wavelength and polarization control may be combined toallow integrating fluorescent molecule orientation information with twoor more types of fluorescent molecules. For example, there may be two ormore different wavelength (color) florescent molecules, each color couldbe specifically oriented to a unique region on biological molecules in arepeating pattern ensemble. When the biological molecules make aconformational change, angular orientation changes could be measuredproviding unique information for each region labeled. This is possiblewith single or multiple molecule applications.

FIG. 27 illustrates a top plan view of one example of an optical fiberlaunch system 2700 utilizing an optical fiber 2710 and an optical fiber2720 aligned via an optical fiber mounting element 2725 such that EMRfrom their terminal ends is directed substantially parallel to eachother through sample slide 2730. The parallel EMR totally internallyreflects causing overlapping evanescent fields 2735 alternating from theopposing opposite surface of sample slide 2730 and the top surface ofsample slide 2730. The EMR from each optical fiber may have similar ordifferent characteristics (e.g., wavelength and/or polarization).

FIG. 28 illustrates a top plan view of another example of an opticalfiber launch system 2800 utilizing an optical fiber 2810 and an opticalfiber 2820 aligned such that EMR from their terminal ends is directedsubstantially perpendicular to each other through sample slide 2830.Optical fiber 2810 is positioned via optical fiber mounting element 2835and directs EMR through sample slide 2830 to an opposing surface ofsample slide 2830 at an incident angle that is at least a critical anglefor TIR. Optical fiber 2820 is positioned via optical fiber mountingelement 2840 and directs EMR through sample slide 2830 to an opposingsurface of sample slide 2830 at an incident angle that is at least acritical angle for TIR. Totally internally reflected EMR from opticalfiber 2810 produces evanescent fields 2850. Totally internally reflectedEMR from optical fiber 2820 produces evanescent fields 2855. Evanescentfields 2850 and 2855 overlap at region 2860. The EMR from each opticalfiber may have similar or different characteristics (e.g., wavelengthand/or polarization).

In another embodiment of the present disclosure, a microscope isprovided that includes a system of the present disclosure. A system or amethod of the present disclosure may be used with a variety ofmicroscopes that are currently configured for various desired techniques(e.g., TIR microscopy) or that are not currently configured for thedesired techniques (e.g., TIR microscopy). Examples of microscopesinclude, but are not limited to, an inverted microscope, an uprightmicroscope, stereoscopic microscope, confocal detection microscope, andscanning confocal detection.

In yet another embodiment of the present disclosure, a system formodifying a microscope to perform TIR microscopy is provided. In oneexample, a microscope may be modified to perform TIR microscopy with anoptical fiber microscopy launching system of the present disclosure.Such a system for modifying a microscope may include a sample slidehaving a first side and an opposing side with a surface, and an opticalfiber having a first terminal end with a terminal surface, the opticalfiber being positioned proximate the first side so as to position theoptical fiber to deliver an EMR from the first terminal end to thesurface of the opposing side at a desired incident angle (e.g., at leasta critical angle for TIR). Another example of a system for modifying amicroscope may include a sample slide as described herein configured toreceive an optical fiber at a position to deliver EMR to an opposingsurface of the sample slide at a desired incident angle (e.g., at leasta critical angle for TIR). Yet another example of a system for modifyinga microscope and/or a sample slide may include a mounting element asdescribed herein that is capable of being optically coupled to astandard sample slide. In another example, a system for modifying amicroscope according to the present disclosure may also include afluorescence detection element that can be fit to the microscope so asto view fluorescence of a sample positioned at the sample slide. Anotherexample of a system for modifying a microscope according to the presentdisclosure includes a source of EMR either attached to a second terminalend of an optical fiber or capable of being attached to the secondterminal end, so as to provide EMR to the first terminal end having theterminal surface for delivery to an opposing surface of a sample slide.

Referring to FIGS. 29 to 32, four example systems for modifying amicroscope are illustrated. FIG. 29 illustrates an example system 2900for modifying a microscope. System 2900 includes a sample slide 2910 inoptical communication with a mounting element 2980 having a fiberinsertion portal 2990 according to the present disclosure. A sample maybe placed on a surface of sample slide 2910 as described herein and anoptical fiber optically coupled with optical fiber mounting element 2980at fiber insertion portal 2990. FIG. 30 illustrates an example system3000 for modifying a microscope. System 3000 includes a sample slide3010 in optical communication with a mounting element 3080 having afiber insertion portal 3090 according to the present disclosure. Opticalfiber 3040 is in fiber insertion portal 3090. Optical fiber 3040includes optical coupling 3042 for connecting to optical coupling 3043of EMR source 3048. FIG. 31 illustrates a sample slide 3110 in opticalcommunication with a mounting element 3180 having a fiber insertionportal 3190 according to the present disclosure. Optical fiber 3140 isinserted in fiber insertion portal 3190. Optical fiber 3140 is inoptical communication with EMR source 3148. FIG. 32 illustrates a sampleslide 3210 in optical communication with the optical fiber 3240 with theimplementation of a coupling medium 3294, such as optical coupling oil3294. Each of these examples may also be provided with a fluorescencedetector. The examples in FIGS. 29 to 32 show a mounting element. Asshould be clear from the present disclosure, a rectangular mountingelement may be replaced by another way of positioning an optical fiber,including, but not limited to another optical fiber mounting element(e.g., an optical coupling medium, a fiber insertion portal in thesample slide), a mechanical coupling, and any combinations thereof.

FIG. 33A illustrates a further embodiment of an optical fiber microscopylaunch system 3300. System 3300 includes a first sample slide 3305 andan optical fiber mounting element 3310 having an optical fiber 3315optically coupled thereto. System 3300 also includes a second sampleslide 3320 and a coupling medium 3325. In one example, second sampleslide 3320 may be used to position a sample 3330. Second sample slide3320 may be readily disposable after one or more uses, while firstsample slide 3305 may be continually utilized with subsequent sampleslides. FIG. 33B illustrates second sample slide 3320 brought intooptical communication with first samples slide 3305 such that EMR fromoptical fiber 3315 totally internally reflects at a surface 3340 ofsecond sample slide 3320.

In still another embodiment of the present disclosure, a method ofperforming TIR microscopy is provided. The method includes providing asample slide having a first side, a second side, and a third side, thefirst side having a first surface, the second side having a secondsurface. The method also includes positioning an optical fiber at afirst position, the optical fiber having a terminal end with a terminalsurface. The first position is proximate the third side so as toposition the optical fiber to deliver an EMR from the first terminal endto the second surface at a first incident angle, the first incidentangle being at least a critical angle for TIR at the second surface. Themethod may also include providing a sample in contact with the firstsurface. The method may further include delivering the EMR to the secondsurface and observing a fluorescence from the sample. In thisembodiment, the first, second, and third sides may be different sides orsame sides. The positioning of the optical fiber can be done by any wayset forth in this disclosure.

In a further embodiment of the present disclosure, a method of modifyinga sample slide for TIR microscopy is provided. The sample slide includesa first side and a second side, the second side having a first surface.The method includes positioning an optical fiber at a first position,the optical fiber having a terminal end with a terminal surface. Thefirst position is proximate the first side so as to position the opticalfiber to deliver an EMR from the first terminal end to the first surfaceat a first incident angle, the first incident angle being at least acritical angle for TIR at the second surface. The positioning of theoptical fiber can be done by any way set forth in this disclosure.

In yet a further embodiment of the present disclosure, a method ofmodifying a microscope for TIR microscopy is provided. The methodincludes providing a sample slide having a first side and a second side,the second side having a first surface. The method also includespositioning an optical fiber at a first position, the optical fiberhaving a terminal end with a terminal surface. The first position isproximate the first side so as to position the optical fiber to deliveran EMR from the first terminal end to the first surface at a firstincident angle, the first incident angle being at least a critical anglefor TIR at the first surface. The sample slide is positioned withrespect to the microscope to allow observation of fluorescence of asample positioned at the sample slide. The positioning of the opticalfiber can be done by any way set forth in this disclosure.

One of ordinary skill will recognize that other methods, such as methodsof performing TIR, modifying a sample slide, and/or modifying amicroscope, are clearly supported using the system of the presentdisclosure described in the multiple embodiments above.

Depending on the context of use, the present disclosure may providebenefits over previous microscopy techniques. One potential benefitincludes the ability to perform a desired microscopy technique,relatively inexpensively, on just about any existing microscope withminor modification. The small amount of equipment needed to deliverexcitation and or illumination EMR to a sample slide allows for easyaccess to the sample. Further, alignment of the EMR is fixed to thesample slide so that movement of the sample slide, such as movementduring focusing, does not disturb alignment of the EMR to the sampleslide. Complicated optical equipment is not needed to deliver EMR from asource to the microscope sample stage. The system and method of thepresent disclosure can be used with a variety of microscopes of avariety of configurations, including (but not limited to) those that areinverted and non-inverted configurations. Traditional illuminationtechniques (such as, but not limited to, brightfield, darkfield, phasecontrast, differential interference contrast, confocal detection, andany combinations thereof) can be used to illuminate the sample and viewthrough typical microscope optics. This is due to the fact that an entryarea of EMR into a sample slide can be positioned away from the samplearea. Some previous prism-type techniques for TIR obstructed the samplearea from some methods of illumination. A system of the presentdisclosure can be made to be disposable after any number of uses. Thiscan eliminate the need to clean sample slides and the negative effectsof buildup on sample slide surfaces. For example, a system including asample slide coupled with an optical fiber having a second end with astandard termination could be removably coupled to an EMR source anddisposed of after one or more uses. Another potential benefit of anexample optical fiber launch system of the present disclosure mayinclude the ability to move a sample slide with respect to a microscopeor other external element without impacting the position and/ororientation of a terminal end of an optical fiber with respect to thesample slide, thus keeping the ability to have the same incident angleat a sample surface despite movement of the sample slide (e.g., movementduring focusing).

Exemplary embodiments have been disclosed above and illustrated in theaccompanying drawings. It will be understood by those skilled in the artthat various changes, omissions and additions may be made to that whichis specifically disclosed herein without departing from the spirit andscope of the present invention.

1. A launch system for total internal reflection (TIR) microscopy, thesystem comprising: a first sample slide having a first side and a secondside; an optical fiber mounting element positioned in optical contactwith said first side; a second sample slide having a third side and afourth side; and an optical fiber having a first terminal end with aterminal surface, said optical fiber being optically coupled with saidoptical fiber mounting element so as to position said optical fiber todeliver an electromagnetic radiation from said terminal end through saidfirst side into said first sample slide, such that when said second sideand said third side are brought into optical contact and a sample ispositioned proximate said fourth side, said electromagnetic radiation isdelivered to said fourth side at an incident angle that is at least acritical angle for total internal reflection at an interface of saidfourth side and the sample.
 2. A system according to claim 1, furthercomprising an optical coupling medium, said optical coupling medium forproviding optical contact of said second side and said third.
 3. Asystem according to claim 2, wherein said optical coupling medium ispositioned proximate said third side.
 4. A system according to claim 2,wherein said optical coupling medium is positioned proximate said secondside.
 5. A system according to claim 2, wherein said optical couplingmedium includes a material selected from the group consisting of anoptical adhesive, glycerol, an optical oil, and any combinationsthereof.
 6. A system according to claim 1, wherein the launch system isconfigured to be positioned proximate a microscope, wherein when thesample is positioned proximate said fourth side, the sample is viewableby the microscope at least in part as a result of the electromagneticradiation.
 7. A system according to claim 1, wherein said optical fiberis positioned to deliver a first subset of the electromagnetic radiationto said fourth surface at an incident angle that is at least a criticalangle for total internal reflection and a second subset of theelectromagnetic radiation to said fourth surface at an incident anglethat is less than a critical angle for total internal reflection.
 8. Asystem according to claim 1, wherein said optical fiber mounting elementcomprises a material selected from the group consisting of an opticaladhesive, glycerol, an optical oil, a prism, a lens, an optical grating,a glass, a plastic, sapphire, a metal, and any combinations thereof. 9.A system according to claim 1, wherein said optical fiber mountingelement and said first sample slide are monolithic.
 10. A systemaccording to claim 1, wherein said optical fiber mounting elementincludes a fiber insertion portal having said optical fiber insertedtherein.
 11. A system according to claim 1, wherein said optical fibermounting element is a fiber insertion portal in said first side.
 12. Asystem according to claim 1, wherein said optical fiber mounting elementis configured to allow the position of said optical fiber to beadjusted.
 13. A microscope comprising a system according to claim
 1. 14.A kit for modifying a microscope to perform total internal reflectionmicroscopy, the kit comprising a system according to claim
 1. 15. Alaunch system for total internal reflection (TIR) microscopy, the systemcomprising: a first sample slide having a first side, a second side, anda fiber insertion portal in said first side; and a second sample slidehaving a third side and a fourth side, said fiber insertion portalhaving a fiber insertion axis, the fiber insertion axis configured suchthat when said second side and said third side are brought into opticalcontact and an optical fiber is inserted within said fiber insertionportal, the optical fiber is positioned so to deliver an electromagneticradiation to said fourth side at an incident angle, such that when asample is positioned proximate said fourth side, said incident angle isat least a critical angle for total internal reflection at an interfaceof said fourth side and the sample.
 16. A system according to claim 15,further comprising an optical fiber inserted in said fiber insertionportal.
 17. A system according to claim 15, further comprising anoptical coupling medium, said optical coupling medium for providingoptical contact of said second side and said third.
 18. A systemaccording to claim 17, wherein said optical coupling medium ispositioned proximate said third side.
 19. A system according to claim17, wherein said optical coupling medium is positioned proximate saidsecond side.
 20. A system according to claim 15, wherein the launchsystem is configured to be positioned proximate a microscope, whereinwhen the sample is positioned proximate said fourth side, the sample isviewable by the microscope at least in part as a result of theelectromagnetic radiation.
 21. A system according to claim 15, whereinsaid fiber insertion axis is configured such that when the optical fiberis inserted therein, a first subset of the electromagnetic radiation isdelivered to said fourth surface at an incident angle that is at least acritical angle for total internal reflection and a second subset of theelectromagnetic radiation is delivered to said fourth surface at anincident angle that is less than a critical angle for total internalreflection.
 22. A microscope comprising a system according to claim 15.23. A kit for modifying a microscope to perform total internalreflection microscopy, the kit comprising a system according to claim15.
 24. A total internal reflection (TIR) microscopy launch system fordelivering an electromagnetic radiation to a first sample slide having afirst side and a second side and to a second sample slide having a thirdside and a fourth side, the system comprising: an optical fiber mountingelement configured to be positioned in optical contact with the firstside; and an optical fiber having a first terminal end having a terminalsurface, said optical fiber being optically coupled with said opticalfiber mounting element so as to position said optical fiber to deliveran electromagnetic radiation from said terminal end through said firstside into said first sample slide, such that when said second side andsaid third side are brought into optical contact and a sample ispositioned proximate said fourth side, said electromagnetic radiation isdelivered to said fourth side at an incident angle that is at least acritical angle for total internal reflection at an interface of saidfourth side and the sample.
 25. A system according to claim 24, whereinsaid optical fiber is positioned to deliver a first subset of theelectromagnetic radiation to said fourth surface at an incident anglethat is at least a critical angle for total internal reflection and asecond subset of the electromagnetic radiation to said fourth surface atan incident angle that is less than a critical angle for total internalreflection.
 26. A method of performing total internal reflection (TIR)microscopy, the method comprising: providing a first sample slide havinga first side and a second side; positioning an optical fiber mountingelement in optical contact with said first side; providing a secondsample slide having a third side and a fourth side; positioning a sampleproximate said fourth side; positioning said second side in opticalcontact with said third side; and optically coupling an optical fiberwith the optical fiber mounting element so as to position the opticalfiber to deliver an electromagnetic radiation from the optical fiberthrough said first side into said first sample slide and into saidsecond sample slide to said fourth side at an incident angle that is atleast a critical angle for total internal reflection at an interface ofsaid fourth side and the sample.
 27. A method according to claim 26,further comprising: delivering the electromagnetic radiation; andobserving a fluorescence from the sample.
 28. A method according toclaim 26, further comprising adjusting the optical fiber to a secondposition in the optical fiber mounting element.
 29. A method ofmodifying a first sample slide for total internal reflection (TIR)microscopy, the first sample slide having a first side and a secondside, the method comprising: positioning an optical fiber at a firstposition that is proximate the first side so as to position the opticalfiber to deliver an electromagnetic radiation into the first sampleslide such that when a second sample slide having a third side and afourth side is positioned with the third side in optical contact withthe second side and a sample is positioned proximate the fourth side,the electromagnetic radiation is delivered into the second sample slideto the fourth side at an incident angle that is at least a criticalangle for total internal reflection at an interface of the fourth sideand the sample.
 30. A method according to claim 29, further comprising:optically coupling an optical fiber mounting element with the firstside; and optically coupling a terminal end of the optical fiber withthe optical fiber mounting element.
 31. A method of modifying amicroscope for a total internal reflection (TIR) microscopy technique,the method comprising: providing a first sample slide having a firstside and a second side; positioning an optical fiber mounting element inoptical contact with said first side; providing a second sample slidehaving a third side and a fourth side; positioning a sample proximatesaid fourth side; positioning said second side in optical contact withsaid third side; and optically coupling an optical fiber with theoptical fiber mounting element so as to position the optical fiber todeliver an electromagnetic radiation from the optical fiber through saidfirst side into said first sample slide and into said second sampleslide to said fourth side at an incident angle that is at least acritical angle for total internal reflection at an interface of saidfourth side and the sample.